JP2552714B2 - Multidentate chelating agent consisting of 8-hydroxyquinoline unit - Google Patents

Abstract

The present invention provides linear, cyclic and trifurcate tri- and tetramine backbone multidentate chelating agents based on the 8-hydroxyquinoline chelating unit. The chelating agents may optionally be substituted with a substrate reactive moiety and antibody-metal ion conjugates may be produced for in vivo diagnostic and therapeutic methods.

Description

DETAILED DESCRIPTION OF THE INVENTION FIELD OF THE INVENTION The present invention relates generally to compounds called ligands. The compounds of the invention bind metals very stably.
More specifically, the present invention relates to a ligand capable of binding to a metal with sufficient stability so that the obtained ligand-metal complex remains intact in a living body. Although such complexes have a variety of uses, of particular relevance to the present invention is their use in radiopharmaceutical applications where the metal atom is a radiation emitting or absorbing absorber isotope. . In one embodiment, the present invention relates to a bifunctional ligand molecule that can be bound to a metal by a specific substrate-reactive group having a phenyl ring on a substrate such as an antibody or other protein. The phenyl ring is substituted in the meta or para position with a substrate reactive residue.
The antibody-metal ion conjugate is used for in vivo diagnostic imaging (imagin
g) can be manufactured for use in methods and treatments,
The metal ions then emit cytotoxic radiation.

(Prior Art and Problems to be Solved by the Invention) Although many factors affect the stability of a ligand-metal complex, some of the properties inherent in the metal itself and the molecule of the ligand molecule are involved. Both properties related to the structure are included. Although the properties of the metal atom can vary widely depending on the type of metal, they are generally unaffected by the operation devised in advance in order to enhance the stability of the complex, and as a result, in the technical field described above, Efforts to maximize the stability of the complex will be focused on the structure of the ligand. Ligand molecules known in the art and relevant to the present invention are generally relatively low molecular weight (100-2000 Dalton) organic molecules, either naturally occurring compounds or synthetic materials tailored for a given purpose. May be

Certain design criteria for producing useful synthetic ligands are well known in the art. The nature of the chemical bond formed between the ligand and the metal depends on the donation of electron pairs present on the ligand molecule, or, in terms of molecular orbital method, the packing orbital on the ligand is It can be considered as a molecular orbital formed by combining with an empty orbital on an atom. Therefore, among the atoms in the ligand molecule, those molecules that are directly involved in forming a chemical bond with the metal atom are called donor atoms and are generally composed of Group V and Group VI elements of the Periodic Table Atoms, oxygen atoms, sulfur atoms, phosphorus atoms and arsenic atoms are most commonly used.

An important criterion for designing a synthetic ligand molecule is that the stability of a metal complex formed by a ligand containing two donor atoms in the same molecule has only a single donor atom. It is based on the fact that, in both cases, the metal and donor atom are the same, albeit large, compared to the corresponding complex formed with the separate ligand molecules contained. The second important design criterion is that the stereochemistry of the ligand molecule is that both donor atoms bond to the metal atom without causing significant steric strain in the ligand molecule framework. It is based on the limitation that it must be possible.

Ligand molecules having two or more donor atoms that can be attached to a single metal atom are called chelating agents and the corresponding metal complexes are called chelates. The thermodynamic phenomenon behind the increased stability due to the presence of two or more donor atoms in the same ligand molecule is called chelate activation [see more specifically FACotton and Wilkinson] (G. Wilkinso
n) "Advanced Inorganic Chemistry", 4th Edition, 71-
See page 73, published 1980]. The number of donor atoms present in a given chelating agent depends on the number of the chelating agent's locus (de
The ligand having two donor sites is called a bidentate ligand, and the ligand having three donor sites is called a tridentate ligand. In the above example, the bidentate ligand was described to explain the chelation effect. However, the same effect can be obtained when the chelating agent has a maximum number of ligands that can be obtained by a specific metal atom. Up to the point of agreement, the higher-dentate ligands also apply.

The maximum number of ligands for a metal atom is an intrinsic property of that atom and is related to the number of empty orbitals it has, and thus the number of chemical bonds it can form. For a given metal atom in a given oxidation state, the maximum coordination number is almost uniform and as a rule cannot be changed by changing the nature of the ligand. A metal atom is said to be coordinately saturated when all available empty orbitals are used to donate atoms in the ligand to form a bond. Therefore, in general, the maximum stability of a metal complex is obtained when a complex is formed using a multidentate chelating agent having a sufficient number of ligands to coordinately saturate the metal atom. Can be achieved. Therefore, an object of the present invention is to provide a polydentate chelating agent capable of obtaining a coordination-saturated complex of a radiopharmaceutical useful metal.

Coordinative saturation of most metals of the first and second transition series and Group IIIb metals of particular relevance to the present invention is
Generally achieved with 6 ligands. Therefore, attempts in the art to design chelating agents capable of forming maximally stable complexes with these metals have been directed to developing ligands with 6 donor atoms. In the field of technology, there has been particular interest in developing hexadentate chelators that form very stable complexes with biologically important metal ions (III). Most of the stable ionic (III) complexes presently known are those produced by various microorganisms and naturally occurring chelating agents as part of the mechanism by which microorganisms ingest ions from the outside world. Therefore, the above efforts often involved biomimetic approaches. Such chelating agents are called siderophores, and the most stable of all metal complexes known in the art is enterobactin.
It is an iron (III) complex of a siderophore called terobactin and has the following structure.

The formation constant of the iron-enterobactin complex is
Rris) et al., J. Amer. Chem. Soc., vol. 101, 6097 (1979) reports 10 ⁵² . The binding of enterobactin to iron occurs through the 6 phenolic oxygen atoms present on the 3 catechol residues. Therefore, the overall structure of this chelating agent consists of three identical bidentate binding units, each unit having the structure:

The rest of the molecule functions as a framework, arranging the three bidentate catechol residues so that all three units can stereochemically associate with one iron center.

The second potent siderophore clinically causes iron secretion and is called desferrioxamine B. It has the structure:

The binding of this chelator to iron is through a series of 6 oxygen atoms present on the three hydroxamate bidentate binding units. Each bond unit has the structure:

As with enterobactin, the rest of the molecule acts as a framework and has sufficient steric flexibility to attach all three bidentate hydroxamate residues to the same iron atom.

Attempts in the art to mimic the above siderophore structure have been to produce compounds using a synthetic framework to properly position the natural catecholate and hydroxamate residues to produce the hexadentate chelator. Has been directed to.

Harris et al., A synthetic hexadentate chelator compound 1,3,
Disclosed is 5-N, N ', N "-tris (2,3-dihydroxybenzoyl) triamomethylbenzene (MECAM), in which three catechol residues are linked to the benzene skeleton.

Jain et al., J. Amer. Chem. Soc., 89, 724.
(1967) and Vol. 90, 519 (1968), tris (2-aminoethyl) amine (TR), which is a tridentate tetraamine as a tetradentate chelating agent for metals such as copper and zinc.
The use of EN) is disclosed.

Inger. Chem., 26, 1622 by Rodgers et al.
(1987) and references cited therein disclose the synthesis of a large number of tridentate tricatechol analogs of enterobactin, including those utilizing TREN as a backbone. The compound forms a very stable complex with iron.

US Pat. Nos. 4,181,654 to Weitl et al., 4,
No. 309,305 and No. 4,543,213, each specification describes four 2,
Disclosed are compounds consisting of an azaalkane framework in which the 3-dihydroxybenzoic acid amide (catechol) residues are approximately cyclically or linearly arranged. The dihydroxybenzoyl group may be optionally substituted with a nitro group, a sulfonate group or a pharmacologically acceptable sulfonate salt. This compound is disclosed to be particularly useful in sequestering actinide (IV) ions by forming an octadentate complex. Wittle et al. U.S. Pat. No. 4,442,30
No. 5 relates to low-dentate polybenzamide compounds consisting of two 2,3-dihydroxybenzoyl groups linked by an azaalkane framework. Such tetradentate compounds are also mentioned to be useful for sequestering actinide (IV) ions in both in vivo and in vitro procedures.

Related to the present invention is a technique relating to chelating agents that utilize atoms other than oxygen as electron donors. Polyaminopolycarboxylate chelating agents such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are well known in the art and form stable complexes with a wide range of metals. Much of its utility stems from the fact that they can combine both oxygen and nitrogen donors. The synthesis of bifunctional DTPA and EDTA analogs is well known. Sandberg et al., J. Med. Chem., Vol. 17, 1304.
(1974) discloses the synthesis of bifunctional EDTA derivatives, which have unique protein substrate reactive functional groups, such as paraaminophenyl protein reactive substituents, attached to the methylene carbon of the polyamine backbone. It is characterized by

Further relevant to the present disclosure is 8-hydroxyquinoline, also known as oxine, in which the bidentate binding unit contains both oxygen and nitrogen donor atoms. This compound has a structure represented by the following formula when bonded to the metal atom M.

As is well known in the art, 8-hydroxyquinoline is also known to complex with a wide range of metals and to be used to extract various metal ions from solution. Plueddemann US Patent No. 4,42
1,654 and Scher U.S. Pat. No. 4,500,494
Various substituted oxines are disclosed in each specification. 8-Hydroxyquinoline is known to be particularly useful in forming complexes with Group IIIb metals. Heavy Group IIIb elements, especially the gamma-emitting isotopes gallium-67, indium-111 and thallium-201 and the positron-emitting metal gallium-68 have found wide application in nuclear medicine, so the latter document Are particularly relevant to the present invention. McCaffey (Mc
Afee) et al., J. Nucl. Med., 17: 480 (1976) discloses labeling blood cells with 8-hydroxyquinoline complexes of indium-111 and technetium-99m.
Int. J. Nucl. Med. Biol., 8 by Moerlein et al.
Vol. 277 (1981) discloses various linear and branched 2,3-dihydroxybenzoylamide analogues of enterobactin and their use as radiopharmaceuticals with gallium and indium. The document also discloses the use of indium-111 labeled 8-hydroxyquinoline. Loberg
No. 4,017,596 to Cobalt-5.
7, gallium-67, gallium-68, technetium-99m,
The use of indium-111 and indium-113m chelates of 8-hydroxyquinoline as radiopharmaceutical external imaging agents is disclosed. European Patent Application No. 83,129 to Goedemans et al. Discloses antibodies and antibody fragments labeled with a radionuclide with a bifunctional chelating agent containing 8-hydroxyquinoline. The chelating agent of that document forms a complex with three 8-hydroxyquinoline ligands attached to each metal atom.

Although tris (8-hydroxyquinoline) complexes are satisfactory for use in the above applications, their usefulness as radiopharmaceuticals in vivo is limited. For example, the bidentate 8-hydroxyquinoline ligand has a formation constant of only 10 ^14.5 for gallium, whereas the formation constant of serum protein transferrin is 10 ^23.7. is there. As a result, once in the bloodstream, the complex tends to decompose and release radiometals to transferrin, making the background level of blood radioactivity undesirably high and persistent.

Bull.Chem.Soc.Japan, Hata et al., 45, 477
(1972) discloses azomethyl and azomethine derivatives of 8-hydroxyquinoline-2-carbaldehyde. One of the derivatives is N, N'-bis (8-hydroxy-2-, consisting of two 8-hydroxyquinoline chelating residues bonded to the ethylenediamine skeleton.
Quinolylmethyl) ethylenediamine. The document is
It is suggested that the tetradentate chelating agent has a higher basicity, and as a result, forms a metal complex more stably than oxine.

Related to the present invention is a disclosure showing the use of protein / metal ion conjugates for diagnostic and therapeutic purposes. U.S. Pat. No. 4,454, Gansow et al.
No. 106 discloses the use of monoclonal antibody / metal ion conjugates for in vivo and in vitro radioimaging. Goldenburg (Go
ldenberg) et al., N. Eng. J. Med., 298, 1384-88 (1978).
Discloses a diagnostic imaging procedure in which an antibody to the known tumor-associated antigen carcinoembryonic antigen (CEA) is labeled with ¹³¹ iodine and injected into cancer patients. After 48 hours, the patient is scanned with a gamma-ray scintillation camera and the tumor site is located by the gamma-ray radiation pattern.

Other investigators have disclosed the therapeutic use of antibody / metal ion conjugates to deliver cytotoxic radioisotopes to in vivo tumor deposits. Int.J.Radiation Oncology Biol.P from Order et al.
hys., 12, 277-81 (1986), describes the treatment of hepatocellular carcinoma with an anti-ferritin polyclonal antibody chelated with ⁹⁰ yttrium. Buchsbaum et al. Int. J. Nucl. Med. Biol., Vol. 12,
No. 2, pp. 79-82 (1985) discloses radiolabeling a monoclonal antibody against CEA with ⁸⁸ yttrium, suggesting that it may be used to position and treat colorectal cancer. There is. Nicolotti European Patent Application No. 174,853, published March 19, 1986, discloses conjugates consisting of metal ions and antibody fragments. According to the disclosure of the document, a monoclonal antibody of subclass IgG is treated enzymatically with F
With the exception of the c fragment, the disulfide bond connecting the H chain of the antibody is cleaved. The Fab 'fragment was then linked to a chelator attached to a radionuclide metal ion and used for in vivo diagnostic and therapeutic purposes. Further relevant to the present invention is J. Nucl. M. of Wang et al.
ed., 28, 723 (1987), which relates to a bifunctional bidentate chelating agent derived from xanthurenic acid, a naturally occurring 8-hydroxyquinoline derivative. This chelating agent has the structure:

The carboxyl group is converted into N-hydroxysuccinimidyl ester, which is reacted with the side chain amino group of the protein to attach the bidentate chelating agent to the substrate. The stability of a single bidentate unit is inadequate for in vivo applications, and the use of complexes containing three xanthurenic acid active ester ligands generally leads to deleterious cross-linking reactions between proteins and at the same time. It is expected to cause denaturation of protein substrates.

(Means for Solving the Problems) The present invention has the general formula: (Where D is And a group selected from the group consisting of —CH ₂ —, R ₁ , R ₂ and R ₃ are the same or different, and are a hydrogen atom, a halogen atom,
C _{1 to} C ₃ alkyl group, nitro group, nitroso group, sulfonate group, sulfonate trialkylammonium salt, phenol group, phosphate group, C _{1 to} C ₃ carboxylic acid group,
A group selected from the group consisting of a carboxamide group, a sulfonamide group, a phosphonic acid group and a sulfate group, n is 3
Or an integer of 4, A is a linear, cyclic or trigeminal triamine or tetraamine, wherein the nitrogen atom of each amine is an alkane via two to four carbon atoms between each amine group, A cycloalkane or ortho-substituted phenyl ring is linked by substituents, and all the oxine units present are positioned such that each is capable of binding to a single metal center, and A is also of the formula: Optionally substituted with a methylene carbon moiety by a group of the formula: wherein X is a nitro group in the meta or para position or a substrate-reactive residue and m is an integer from 0 to about 10. ) Concerning the compound shown.

The present invention also provides a compound of the general formula: (In the formula, m is an integer of 0 to about 10, p is an integer of 0 or 1,
q is an integer of 0 or 1, r is an integer of 0 or 1, s is an integer of 2, 3 or 4, t is an integer of 2, 3 or 4, u
Is an integer of 0, 1, 2 or 3 and v is 0, 1, 2 or 3
, U + v is an integer of ₁ , 2 or 3 and S ₁ ,
_{S 2, S 3, S 4} , S 5 and S ₆ are the same or different, -H,
_{-OH, -CH 2 CO 2 H,} -CH 2 CH 2 CO 2 H, and (In the formula, y is an integer of 1 to about 5, S ₇ is an aryl group or C ₁
To C ₂₀ alkyl group) and the following formula and (Wherein S ₈ and S ₉ are the same or different and are C _{1 to} C ₂₀ alkyl groups or aryl groups), and a cyclic substituted or unsubstituted aromatic metal bond residue selected from the group represented by X and X is Bidentate chelating agents represented by nitro groups or substrate-reactive residues in the meta or para positions are also provided.

The present invention provides numerous chelating agents of various sizes, shapes and loci, and there is no single preferred chelating agent of the present invention. Preferred compounds are
Varies according to the characteristics of the metal ion to be bound.

The bifunctional chelating agent of the present invention binds a metal (including a radioactive metal ion) to various substrate molecules (protein, glycoprotein, peptide, polyamino acid, lipid, carbohydrate,
Suitable for attachment to polysaccharides, nucleosides, nucleotides, nucleic acids, drugs, inhibitors and whole cells).

In one embodiment of the invention, antibody conjugates and antibody-metal ion conjugates containing bifunctional chelating agents are provided. The present invention further provides in vivo diagnostic imaging utilizing radiation emitting and absorbing metal ions. According to yet another aspect of the invention there is provided a therapeutic method for the treatment of a condition such as cancer. In that case, the cytotoxic radiation emitting nuclide is linked to the anti-tumor associated antigen antibody by the chelating agent of the invention. Then, when the antibody-radionuclide conjugate according to the present invention is introduced into the body of a patient, the cytotoxic radionuclide is selectively directed to cells having a tumor-associated antigen. The compounds of the present invention are also useful in in vivo therapeutic methods where it is desired to remove metal ions, especially radioactive metal ions, from the body, for example where the patient is radioactively contaminated.

(Outline of Invention) Hereinafter, the present invention will be described in more detail.

The present invention relates to polydentate chelating agents based on 8-hydroxyquinoline units. The chelating agents of the present invention may be substituted or may be in various forms and may be bifunctional consisting of a substrate reactive group containing a substrate reactive residue in addition to the chelating group. . The bifunctional compounds of the present invention may be covalently linked to antibodies, other proteins and the like, and are particularly useful for in vivo diagnostic and therapeutic purposes.

The present invention also provides triamine triamine tris- (2-
Also provided are bifunctional polydentate chelating agents based on aminoethyl) amine (TREN). The above tetraamine has the following formula: (In the formula, X is a nitro group or a substrate-reactive residue, and m is 0 to
Substrate-reactive groups, which are integers of about 10), may be substituted at the methylene carbon moiety. Jane Am J. Am
er.Chem.Soc., 89, 724 (1967) and 90, 519 (1
968), the original tetraamine itself can function as a tetradentate chelating agent for metals such as copper and zinc. Bifunctional analogs of TREN may be covalently attached to substrates, including antibodies and other proteins, via substrate reactive residues.

Higher locus bifunctional chelators can also be obtained by further derivatization of the TREN backbone. Sim et al., Inorg. Chem., 17: 1288 (1978) and Wilson et al., J. Amer. Chem. Soc., 90, 604.
1 (1968), monofunctional T
Examples of compounds derived from the REN skeleton include imines formed by Schiff base condensation with salicylaldehyde or pyridine-2-carboxaldehyde. As another example, Rogers et al., Inorg. Chem., 26,
Amides can be generated by acylating a primary amine with a chelating residue according to the method described in 1622 (1987). TREN a very wide range of metal binding residues
It can be replaced on the arm. Such metal binding residues include hydrogen atoms, hydroxyl groups, carboxymethyl groups, carboxyethyl groups and hydroxamate groups as well as aromatic and heterocyclic metal binding residues. The aromatic and heterocyclic metal bond residues may be unsubstituted or substituted at various positions on the ring, such as 8-hydroxyquinoline, with various substituents such as hydroxyl and sulfonate groups. Good. The arms of the TREN may be extended so that each arm contains as many as 4 methylene carbons, but in general each arm contains only 2 or 3 methylene carbons for reasons of bond stability. Is preferred.

Although certain substituents and residues of the chelating compounds of this invention are generally preferred, the particular structure of the compounds of this invention will vary widely depending on the nature of the substrate and metal ion being attached. Nevertheless, certain general principles can be applied to prepare the compounds of the present invention for a particular purpose of use. For example, certain metal ions, such as actinide and lanthanide series metals, have large radii and therefore preferably have a large number of ligands. Preferred chelating agents for binding such ions are large compounds with large loci.
As another example, the exact position of a particular amino acid residue in the structure of a protein (either on or near the active site of an enzyme, or on or near the recognition region of an antibody) may result in another substrate-reactive residue on the chelator. You may need to choose a group.

Most metal ions of interest are 6-ligands or less, and thus are generally better bound by the hexadentate compounds of the invention (n = 3). in spite of,
Some metal ions may have a higher number of ligands, for example indium 7 ligands and yttrium 8 ligands. As a result, octadentate chelating compounds (n = 4) may be particularly advantageous. However, due to steric limitations in arranging five or more oxine substituents around the metal ion, compounds with higher loci (n = 5) consistent with the teachings of the present invention. It is not clear whether (or more) will bring any particular advantage. While such steric constraints are of great interest to the octadentate compounds of the invention, they may limit substitution of the oxine ring on such compounds. Nevertheless, particular advantages may be obtained by linking two or more compounds of the invention in such a way that the resulting macrostructure can simultaneously bind two or more metal ions. It is thought to be impossible.

The 8-hydroxyquinoline binding functionality is preferably utilized in the quinoline compounds of the present invention. Nevertheless, it is believed that 8-mercaptoquinoline analogs may also be suitable for use in accordance with the present invention.
The nitrogen atom and oxygen (or sulfur) atom on the oxine unit fulfill the function of binding to the metal atom, but the other atom of the double ring may be substituted. Generally, such substitutions do not affect the metal binding properties of the compound unless it sterically hinders binding. However, the substitutions affect properties such as solubility that are critical to utilizing the compounds of the invention for various applications. The unsubstituted compounds of the present invention tend to exhibit relatively low solubility in aqueous solution. Therefore, it is preferable to replace the oxine unit with a hydrophilic residue such as sulfonate, which can easily increase the solubility of the compound of the present invention. According to these observations, at least one of R ₁ , R ₂ and R ₃ is a sulfonate, and R ₁ , R ₂ and R ₃ are substituted so that they are substituted at the 4-position, most preferably the 5-position on the oxine ring. It is preferable to select it.

The skeleton A of the compounds of the present invention is a linear, cyclic or trifurcated triamine or tetraamine, wherein the nitrogen atom of each amine is 2 to 4 between the nitrogen atoms of each amine.
Linked via an alkane, cycloalkane, or ortho-substituted phenyl ring substituent via 4 carbon atoms and also positioned such that all the oxine units present are each capable of bonding to a single metal center. ing.
The backbone structure is limited by steric and thermodynamic constraints on these polyamines having 2-4 carbon atoms between the amine groups. The small skeleton tends to be sterically constrained and thus unable to position the oxine unit for attachment to the metal center. Skeletons with more than four carbon atoms between amine groups result in a large amount of disorder (entropy) exhibited in long oxine arms,
It tends to exhibit low metal binding efficiency. It is therefore preferred that the compounds of the present invention have the amine nitrogen atom linked with an alkane substituent having 2 to 4 carbon atoms, most preferably 2 or 3 carbon atoms, between the amine groups. . Particularly preferable skeleton structures are three of diethylenetriamine, 1,4,7-triazacyclononane and tris (2-aminoethyl) amine (TREN) represented by the following formula.

and Particularly preferred compounds of the present invention include the following compounds.

and Substrate Reactive Residues The compounds of the present invention have the formula: (In the formula, m is 0 to about 10, preferably 1,) and may optionally include a substrate-reactive group. It can be seen that when X is a nitro group in the above formula, it is necessary to further convert the substrate-reactive group before reacting with the substrate. Examples of the substrate-reactive group for X include those selected from the following group.

-NH ₂ (amino) -NN ⁺ (diazonium) -NCS (isothiocyanate) -NCO (isocyanate) -NHNH ₂ (hydrazine) -NHCONHNH ₂ (semicarbazide) -NHCSNHNH ₂ (thiosemialbazide) -NHCOCH ₂ Cl (chloroacetamide) ) -NHCOCH ₂ Br (bromoacetamide) -NHCOCH ₂ I (iodoacetamide) -N ₃ (azide) -CO ₂ H (carboxylate) -NHCONH (CH ₂ ) _w NH ₂ (aminoalkylurea) -NHCSNH (CH ₂ ) _w NH ₂ (aminoalkyl _{thioureas) -NHCONH (CH 2) w CO} 2 H ( carboxyalkyl _{urea) -NHCSNH (CH 2) w CO} 2 H ( carboxyalkylthiourea) and (In the formula, Y is a halogen atom selected from the group consisting of Cl, Br and F, Z is a group selected from the group consisting of Cl, Br, F, OH and OCH ₃ , and w is an integer of 1 to about 10. in a) particularly preferred substrate reactive residue, -NH ₂ where X is substituted at the para position, -NCS, -NHCOCH ₂ Br and -NHCSNH (CH _{2) 2}
It is a group selected from the group consisting of NH ₂ . Preferred substrate-reactive residues include those that can react with sugar chains present on some proteins, where it is desirable to avoid direct conjugation of protein substrates to side chain amino acids. Since such sugar chains are generally inactive, it is often necessary to derivatize or oxidize them to increase their reactivity. Preferred substrate reactive residues for use in binding to glycosylated proteins include -NHNH ₂ and -NHCSNHNH _2.

Metal Ions The metal ions chelated in accordance with the present invention include gamma-emitting isotopes useful in diagnostic scintigraphy. Other suitable gamma emitters include ⁶⁷ gallium, ⁶⁸ gallium and ^99m technetium, although ¹¹¹ indium with a half-life of 2.8 days is particularly useful. The compounds according to the invention may be chelated to beta radiation emitters which are useful in radiotherapy as cytotoxic agents. Such emitters include ⁴⁶ scandium, ⁴⁷ scandium, ⁴⁸ scandium, ⁶⁷ copper, ⁷² gallium, ⁷³ gallium, ⁹⁰ yttrium,
⁹⁷ ruthenium, ¹⁰⁰ palladium, ^101m rhodium, ¹⁰⁹ palladium, ¹⁵³ samarium, ¹⁸⁶ rhenium, ¹⁸⁸ rhenium,
Includes ¹⁸⁹ rhenium, ¹⁹⁸ gold, ²¹² radium and ²¹² lead.

The chelating agents of the present invention also include alpha radiation emitting materials such as ²¹² bismuth, positron emitters such as ⁶⁸ gallium and ⁸⁹ zirconium, lanthanide series elements such as terbium and europium and transition series elements such as ruthenium. It may also be used to bind to polar elements and paramagnetic substances such as gadolinium and iron.

Complexation of Metal Ions Methods for forming chelating agent / metal ion conjugates are well known to those skilled in the art. Generally, a chelator / metal ion complex may be formed by incubating the chelator / substrate conjugate with a metal ion in a buffer solution in which the conjugate is stable. Suitable buffers include those with weak metal binding properties such as citrate buffer, acetate buffer and glycine. Appropriate concentrations, temperatures and pH's may be selected by those skilled in the art so that the metal ion binds to the chelating functional group rather than the weak metal binding site on the substrate. It is especially desirable that all solutions be free of metallic impurities. After incubation for a suitable time, unbound metal ions may be separated from the substrate / chelating agent / metal ion conjugate by procedures such as gel filtration if necessary.

Substrate Reactive Functionality Substrate reactive residues of the invention are those residues that are capable of specifically binding to at least one functional group present in a substrate molecule (which may be a biologically active substrate). Become.
When the substrate is a protein, such residues may be reactive with the side chain groups of the amino acids that make up the polypeptide backbone. Such side chain groups include the carboxyl groups of aspartic acid and glutamic acid residues, the amino groups of lysine residues, the aromatic groups of tyrosine and histidine residues and the sulfhydryl groups of cysteine residues.

The side chain carboxyl groups provided by the substrate, such as the polypeptide backbone, may be reacted with the amine substrate reactive groups of the compounds of the invention by a soluble carbodiimide reaction. The side chain amino group provided by the substrate may be reacted with an isothiocyanate, isocyanate or halotriazine derivative of the invention to attach a chelating agent to the polypeptide. Alternatively, a bifunctional agent such as a dialdehyde or imide ester may attach the side chain amino group present on the substrate to the compound of the invention having an amine substrate reactive group. The aromatic group provided by the substrate may be attached to the chelating agent of the present invention by a diazonium derivative. Sulfhydryl groups present on the substrate molecule may be reacted with haloalkyl substrate reactive groups such as maleimide or iodoacetamide. Free sulfhydryl groups suitable for such reactions may be obtained from disulfide bonds of immunoglobulin proteins or introduced by chemical derivation. Binding to a sulfhydryl group generated in the heavy chain region of immunoglobulin does not interfere with the antibody binding site of immunoglobulin,
The antibody may render it unable to activate complement.

When the substrate is a glycosylated protein, an alternative to generating a bond to the compounds of the invention via the polypeptide backbone is described by Rodwell et al. In US Pat. No. 4,671,958. According to the invention, to form covalent bonds with side chain carbohydrates of glycoproteins. Therefore, the side chain carbohydrate of an antibody selectively oxidizes it to form an aldehyde, which is then reacted with an amine substrate reactive group to form a Schiff base, or a hydrazine, semicarbazide or thiosemicarbazide substrate. Reacting with a reactive group produces the corresponding hydrazone, semicarbazone or thiosemicarbazone bond. These same methods may also be used to attach the bifunctional chelating agents of the present invention to non-proteinaceous substrates such as carbohydrates and polysaccharides.

Another substrate-reactive residue useful for coupling to carbohydrates and polysaccharides without the need for prior oxidation is a dihydroxyboryl residue such as those present in the meta- (dihydroxyboryl) phenylthiourea derivatives of the invention. It is a base.
This residue reacts with a substrate containing 1,2-cis-diol to form a 5-membered borate ester. It is therefore useful for carbohydrates, polysaccharides and glycoproteins containing this group. The dihydroxyboryl derivatives can also be used to attach the chelating agents of the invention to ribonucleosides, ribonucleotides and ribonucleic acids. This is because Biochemis of Rosenberg et al.
This is because ribose contains 1,2-cis-diol at the 2 ', 3' position as disclosed in Try, Vol. 11, 3623-28 (1972). Deoxyribonucleotides and DNA cannot be attached to the chelating agents of the present invention by this method since they do not have a 3'hydroxyl group. However, these substrates were coupled to the isothiocyanate derivative of the chelating agent by first generating the allylamine derivative of the deoxyribonucleotide, as disclosed in European Patent Application No. 97,373 to Engelhardt et al. Can be made.

When the substrate to be attached to the chelating agent of the present invention is whole cells, polypeptide-reactive residues or carbohydrate-reactive residues can be used. Hwan
g) and Wase's Biochim.Biophys.Acta, 5
Volume 12, 54-71 (1978), Sandberg (Sundberg)
Bifunctional ED by J. Med. Chem., Vol. 17, et al., 1304 (1974).
The use of the TA chelator diazonium derivative for indium-111 labeling of red blood cells and platelets is disclosed. Dihydroxyboryl residues are reactive against various bacteria, viruses and microorganisms [Zittl
e) Advan. Enzyme., 12: 493 (1951) and Burnett et al., Biochem. Biophys.Res.Comm., 96, 1
57-62 (1980)].

According to the present invention, the substrate-reactive residue has an amino group (-NH
_2), diazonium group (-NH ^+), isothiocyanate group (-NCS), isocyanate group (-NCO), hydrazine group (-NHNH _2), semicarbazide group (-NHCONHNH _2), thiosemicarbazide group (-NHCSNHNH ₂₎ , A haloacetamide group (-NHCOCH ₂ X) (including chloroacetamide, bromoacetamide and iodoacetamide), an azide group (-N ₃ ), a carboxylate group (-CO ₂ H), an aminoalkylurea (-NHCONH (CH ₂ ) _w NH _2), aminoalkyl thiourea _{(-NHCSNH (CH 2) w NH} 2), carboxyalkyl urea _{(-NHCONH (CH 2) w CO} 2 H) and carboxyalkylthiourea _{(-NHCSNH (CH 2) w CO} 2 H) (where w is about 1
To about 10 integer), maleimide, halo-triazine (chlorotriazines, bromo triazine and iodo triazine) and meta - (dihydroxy Helsingborg) phenylthiourea _{(-NHCSNHC 6 H 4 B (OH} ) 2) are included. Other reactive residues suitable for attaching the chelating agent to the substrate include disulfides, nitrenes, sulfonamides, carbodiimides, sulfonyl chlorides, benzimidates, -CO
CH ₃ and -SO ₃ H include. The preferred substrate-reactive residues for a particular application of the invention will be determined by the nature of the substrate and its susceptibility to loss of biological activity as a result of forming certain bonds. The formation of a certain bond involves the chemical conversion of the substrate-reactive residue X into a conjugated form (hereinafter referred to as a residue of X).

The reactive residues of the present invention are oriented in the meta position, preferably the para position, on the phenyl group, which phenyl group is attached to the chelating agent skeleton of the present invention by an aliphatic spacer group. The spacer group may consist of 1 to about 10 carbon atoms and may be a straight chain or branched chain alkyl group or a substituted alkyl group, although such branched chain or substituent group is a metal. It should not interfere with the binding site or substrate-reactive group. Nevertheless, linear alkyl linkers are preferred, and C ₁ alkyl linkers are particularly preferred.

Substrates Useful in the Invention Substrate molecules that may be reacted with the chelating agents of the invention include proteins, glycoproteins, peptides, polyamino acids, lipids, carbohydrates, polysaccharides, nucleosides, nucleotides, nucleic acids, pharmaceuticals, inhibitors and whole molecules. Contains cells. Suitable proteins include immunoglobulins, antigens, enzymes, components of the blood coagulation / anticoagulation system and various biochemically active molecules and receptors. Such proteins may be derived, for example, from genetically engineered cells. According to one embodiment of the invention, bifunctional chelating agents may be used to bind various types of antibodies including IgA, IgD, IgE, IgG and IgM. Antibodies are directed against various antigenic determinants associated with tumors, histocompatibility and other cell surface antigens, bacteria, molds, viruses, enzymes, toxins, pharmaceuticals and other biologically active molecules. You may Tumor-associated antigens with which the antibody can specifically react include Zalcbe (Zalcbe).
rg) and McKenzie et al., J. Clin. Oncol.
ogy, Vol. 3, 876-82 (1985), including carcinoembryonic antigen (CEA), mucins such as TAG-72, human milk fat globular antigen.
antigens) and receptors such as the IL-2 receptor and transferrin receptor. Such antibodies may be monoclonal or polyclonal, and may be derived from Morrison et al., Proc. Nat. A.
cad.Sci.USA, Vol. 81, 6851-55 (1984).

Fragments of antibody molecules may also be attached, such as Fab, Fa
b ', F (ab') ₂ fragments are included. Nicolotti European Patent Application No. 174,85 published March 19, 1986
No. 3 discloses a method in which whole antibody is treated to carry out site-specific cleavage of two H chains to remove the Fc portion at the carboxyl terminus of the H chains.

The substrate is reacted with the substrate-reactive site of the chelating agent according to the invention described above. Each substrate may optionally be associated with more than one chelating agent. However, the maximum extent of substitution on a substrate such as a protein is limited by the nature of the sugar chain of the protein or the number and position of reactive side chain amino acids on the protein molecule. When it is desired that the binding protein retain its biological activity, such as in the case of antibodies, the degree of substitution depends on the nature of the target sugar chain or amino acid residue in both the primary and tertiary sequences of the protein. And location and the extent to which they participate in the antigen binding site.

Other substrates that can be used in the present invention include polysaccharide matrices for extracting metals from metalloproteins and other metal-containing materials when derivatized with the chelating agents of the present invention. Means, and Biochemistry of Porath and Olin, 22
Vol., 1621-30 (1983), to provide a means for performing affinity chromatography of proteins. Nucleic acids conjugated with chelating agents of the present invention can be used to monitor any nucleic acid hybridization reaction, as described in European Patent Application No. 97,373 to Engelhard et al. A bifunctional chelator coupled to a drug can be used to track uptake of the drug into tissues, an example of which is Sandberg et al., J. Med., For imaging tumors.
Chem., Vol. 17, 1304 (1974), in which bleomycin which is an antibiotic drug is bound to the bifunctional EDTA derivative is used [J. Me of DeRiemer et al.
d.Chem., Vol. 22, 1019-23 (1979); Goodwin
Win) et al., J. Nucl. Med., Vol. 22, 787-92 (1981)].
Whole cells such as erythrocytes and platelets are described by Fang and Weiss in Biochim. Biophys. Acta, 512, 54-71 (1978).
Radiolabeled metal labeling by coupling to a bifunctional chelating agent as described in. Such labeled cells can be used to detect areas of abnormal accumulation in the body. It is also envisioned that the compounds of the present invention will be conjugated to low molecular weight substances which themselves undergo specific binding reactions with macromolecular biological molecules. Archer Biochem. Biophys., 231, 477-8 by Haner et al.
6 (1984) discloses a method for coupling EDTA to p-aminobenzamide. This p-aminobenzamide is a specific inhibitor of trypsin, which binds strongly at the active site, providing the affinity label used to probe that site.
J. Ame of Schultz and Dervan
r. Chem. Soc., 105, 7748-50 (1983), describes the N-methylpyrrole tripeptide distamycin (distam
The sequence-specific double-strand breakage of DNA by the iron complex of the conjugate obtained by binding EDTA to ycin) is disclosed, wherein the tripeptide binds to DNA in a sequence-specific manner.

Next, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.

The following examples illustrate the method of synthesis of various chelating agents according to the present invention. Examples 1-5 are 2-carboxy-8-hydroxyquinoline and its sulfonate analog 2-carboxy-5-sulfonate-8-.
A method of synthesizing hydroxyquinoline is described and these compounds are used to prepare the polydentate chelating compounds of the present invention.

Examples 6-8 describe synthetic methods for various hexadentate chelating agents according to the present invention. Example 6 is a tridentate chelating agent, tris-N- (2-aminoethyl- [8-hydroxyquinoline-2-carboxamide]).
The synthesis of amines is described. Example 7 describes the synthesis of the cyclic hexadentate chelator, N, N ', N "-tris (hydroxyquinoline-2-carboxamido) -1,4,7-triazacyclononane. Describes the synthesis of N ¹ , N ⁴ , N ⁷ (tris- (8-hydroxyquinoline-2-carboxamide)) diethylenetriamine, a linear hexadentate chelating agent of the present invention.

Examples 9-13 describe the synthesis of bifunctional TREN molecules. Example 13, N ⁴ - [2- Amino-ethyl-2- (4-
Nitrobenzyl)] diethylenetriamine tetrahydrochloride is described, which is the bifunctional TREN tetrahydrochloride. The bifunctional TREN molecule of Example 13 can be used as a bifunctional chelating agent itself or modified with various chelating residues to produce other chelating agents.

Examples 14-16 describe the synthesis of various bifunctional analogs of the tridentate hexadentate chelator of Example 6. The bifunctional analog is synthesized from the bifunctional TREN tetrahydrochloride salt obtained according to Example 13. Such bifunctional chelating agents can attach metal ions to reactive residues including antibodies and other proteins. Example 14, N ⁴ - [2- Amino-ethyl-2- (4-nitrobenzyl)] tris -N-
The synthesis of (8-hydroxyquinoline-2-carboxamide) diethylenetriamine is described. Example 15, by converting the nitro residue of the compound of Example 14 the amino substrate reactive group, N ⁴ - [2-Amino-ethyl-2- (4-
Aminobenzyl)] tris-N- (8-hydroxyquinoline-2-carboxamido) diethylenetriamine dihydrochloride synthesis is described. Example 16, by converting the amino acid residues of the compound of Example 15 in isothiocyanate substrate reactive group, N ⁴ - [2-Amino-ethyl-2- (4-
The synthesis of isothiocyanatebenzyl)] tris-N- (8-hydroxyquinoline-2-carboxamido) diethylenetriamine hydrochloride is described.

Example 17 is a sulfonate analog of the tridentate hexadentate chelator of Example 6, Tris-N- (2-aminoethyl- [8-hydroxyquinoline-5-sulfonate-2.
-Carboxamide]) amines. Example 18 is
The chelating agent Tris-N- (2-
The synthesis of an indium complex of aminoethyl- [8-hydroxyquinoline-2-carboxamido]) amine is described. Example 19 describes the preparation of a conjugate between the bifunctional chelator of Example 16 and a monoclonal antibody. Example 20 shows indium with the antibody conjugate obtained in Example 19.
The preparation of the radioactive metal ion complex of 111 is described.

Example 1 (Preparation of 8-hydroxyquinoline-N-oxide) In this example, 8-hydroxyquinoline-N-oxide was produced from 8-hydroxyquinoline. CHCl ₃ (550
A stirred solution of 8-hydroxyquinoline (25.00 g, 172.2 mmol) in 3 ml) was cooled to 0 ° C and 3-chloroperoxybenzoic acid (40.00 g, 80% technical grade x 0.23
1 mmol = 0.185 mmol) was added slowly over 3 minutes. The solution was kept at 0 ° C. and stirred for 3 hours. During this time the 3-chlorobenzoic acid by-product precipitated. This 3-
Except chlorobenzoic acid by filtration, the filtrate was concentrated to dryness orange was triturated with remaining solid _{2% NH 4 OH (2 ×} 200ml). The solid was isolated on a frit and washed with water. The solid was dried under vacuum and recrystallized twice from 10: 1 hexane-acetone to give 8-hydroxyquinoline-N.
-Oxide was obtained as pale yellow needles. Melting point: 138-39
C (21.36 g, 132.7 mmol, yield 77%). ¹ H NMR (CDCl ₃ , TMS internal standard) δ: 7.05 (d, J = 7.5H)
z, 1H), 7.22 (d, J = 10.5Hz, 1H), 7.25 (t, J
= 7.5Hz, 1H), 7.48 (t, J = 7.4Hz, 1H), 7.79
(D, J = 9 Hz, 1H), 8.25 (d, J = 6.0 Hz, 1H) (phenol protons were not found) ¹³ C NMR δ: 114.67, 116.65, 120.26, 129.54, 130.4
0, 132.08, 134.34, 153.79 Mass spectrum (C ₉ H ₇ O ₂ N = 161.16 g / mol, m /
e): 161 (M ⁺ , base peak), 116, 89, 63 IR spectrum: 2248m, 1560s, 1530m, 1470m, 1280s, 11
89m, 1156m, 1049m, 816s Example 2 (Preparation of 8-hydroxyquinoline-N-methoxymethylsulfate) In this Example, 8-hydroxyquinoline-N-methoxymethylsulfate was prepared. A stirred suspension of 8-hydroxyquinoline-N-oxide (37.0 g, 229.6 mmol) obtained in Example 1 in CCl ₄ (750 ml) was refluxed under a nitrogen atmosphere. When the solution was refluxed, the rest of the 8-hydroxyquinoline moved into the solution, producing a yellow homogeneous solution. If the mixture was not homogeneous, additional CCl ₄ was added. Dimethyl sulfate (66.65 g, 528.4 mmol)
Was added to the reflux solution, and the mixture was stirred for 8 hours while refluxing. The desired product floated to the top of the reaction mixture as the reaction proceeded. The reaction stopped when no more material was separated from the mixture. The reaction mixture was cooled to room temperature and the CCl ₄ layer was decanted off. The remaining dark red tar was washed with dry diethyl ether and dried under vacuum for 48 hours to give 8-hydroxyquinoline-N-methoxymethylsulfate as a microcrystalline dark orange solid (58.91 g, 89% yield). %). This material was extremely hygroscopic, so no further purification steps were performed. ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 3.45 (s, 3 H),
4.46 (s, 3H), 7.67 (d, J = 9.00Hz, 1H), 7.89
(T, J = 9.00Hz, 1H), 7.94 (d, J = 6.00Hz, 1
H), 8.14 (t, J = 6.00Hz, 1H), 9.24 (d, J = 9.0
0Hz, 1H), 9.76 (d, J = 6.00Hz, 1H) Mass spectrum (C ₁₀ H ₁₀ O ₂ N ⁺ SO ₄ CH ₃ = 287.28 g / mol, positive fast atom impact, M ⁺ , base peak , Parent cation): 176, 190, 226, 256, 270, 289 Example 3 (Preparation of 2-cyano-8-hydroxyquinoline) In this example, 2-cyano-8-
Hydroxyquinoline was prepared. Sodium cyanide (30.11g, 614.5mmol) was dissolved in water (150ml) and iced.
/ NaCl bath cooled to 0 ° C. 8-prepared in Example 2
Hydroxyquinoline-N-methoxymethyl sulfate (58.91 g, 205.1 mmol) was dissolved in water (250 ml),
Gravity filtration was performed in the P5 filter paper. The 8-hydroxyquinoline-N-methoxymethylsulfate solution was then added to the stirred cyanide solution at 0 ° C over 2 hours. The mixture was stirred for another hour. The pH of the solution was lowered to 4.5 with concentrated acetic acid. This operation precipitated the desired material as a gray solid. This material was isolated on a coarse frit, washed well with water and dried under vacuum for 24 hours. This substance
Recrystallized from 10: 1 hexane-acetone to give 2-cyano-
8-Hydroxyquinoline (25.00 g, 146.9 mmol, 72% yield) was obtained as light brown needle crystals. Melting point: 132-43 ° C. ¹ H NMR (CDCl ₃ , TMS internal standard) δ: 7.28 (d, J = 6.0H
z, 1H), 7.41 (d, J = 7.0Hz, 1H), 7.63 (t, J =
6.0Hz, 1H), 7.71 (d, J = 7.0Hz, 1H), 7.9 (s, 1
H), 8.30 (d, J = 8.0 Hz, 1H) Mass spectrum (C ₁₀ H ₆ O ₂ N = 170.17 g / mol, m /
e): 171 (M ⁺ H, base peak), 162, 146, 188 (M ⁺ + N)
H ₃ ) Example 4 (Preparation of 2-carboxy-8-hydroxyquinoline) 2-cyano-8-hydroxyquinoline (25.00 g, 146.9 mmol) obtained in Example 3 was stirred with 3N NaOH (water 37
45.01 g in 5 ml). The solution was refluxed for 5 hours. pH
When the paper no longer detected base at the top of the condenser, the reaction was stopped and allowed to cool to room temperature. The pH of the reaction mixture was lowered to 4.5 with 5N HCl. The desired product was then extracted into ethyl acetate (2). The ethyl acetate solution was condensed to dryness and collected as a 2 g fraction in methanol (200 ml) and separated on a Sephadex LH-20 column (75
0 g, eluted with methanol) for further purification. The main fraction was isolated from the column and recrystallized from water to give 2-carboxy-8-hydroxyquinoline (20.10 g, 10
6.3 mmol, 72% yield) were obtained as bright yellow crystals. Melting point: 215-216 ° C. ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 7.25 (d, J = 9.
0Hz, 1H), 7.54 (d, J = 7.50Hz, 1H), 7.65 (t,
J = 7.4Hz, 1H), 8.18 (d, J = 9.0Hz, 1H), 8.58
(D, J = 9.0 Hz, 1H), 10.23 (broad s, 1H) ¹³ C NMR (δ): 111.98, 117.56, 119.92, 129.89, 130.
42, 136.43, 138.29, 144.23, 153.80, 165.10 Mass spectrum (C ₁₀ H ₇ O ₃ N = 189.17 g / mol, m /
e, M ⁺ H, base peak): 190, 172, 162, 143, 116, 10
4,89 Example 5 (2-carboxy-5-sulfonate 8 Preparation of hydroxyquinoline) 2-carboxy-8-hydroxyquinoline was obtained in Example 4 (1.00 g, 5.3 mmol) of concentrated H ₂ SO ₄ (the 20 ml) and heated to 100 ° C. for 4.5 hours with stirring. The mixture was left to cool to room temperature and left for 48 hours to slowly complete the reaction. Water (150 ml) was added slowly to the solution and the resulting mixture was cooled to 5 ° C. Over the next 48 hours the desired material crystallizes from solution and becomes 2-carboxy-5-
Sulfonate-8-hydroxyquinoline was obtained as dark orange hairy crystals (0.99 g, 3.7 mmol, yield 70).
%). Melting point:> 350 ° C. ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 7.11 (d, J = 9.
0Hz, 1H), 8.01 (d, J = 7.5Hz, 1H), 8.22 (d, J
= 9.0 Hz, 1 H), 9.42 (d, J = 9.0 Hz, 1 H) (phenolic carboxylic acid proton and sulfonic acid proton were not found) ¹³ C NMR (δ): 110.02, 120.02, 126.64, 128.82, 135 .
22,136.68,138.52,144.09,154.52,165.33 mass spectra _{(C 10 H 7 O 6 NS} = 269.23g / mol, m / e, M ⁺ H
Parent ion peak): 270, 226, 190, 146, 130 Example 6 (Preparation of tris-N- (2-aminoethyl- [8-hydroxyquinoline-2-carboxamido]) amine) The 2-carboxy-8-hydroxyquinoline obtained in Example 4 (1.00 g, 5.3 mmol) was dissolved in dry tetrahydrofuran (THF) (100 ml). N-
Hydroxysuccinimide (0.66 g, 5.7 mmol) and 1,3-dicyclohexylcarbodiimide (1.16 g, 5.7 mmol)
Mmol) was added. The reaction vessel was sealed with a septum and stirred at room temperature for 24 hours. The precipitated 1,3-dicyclohexylurea byproduct was removed from the solution by filtration. Tris- (2-aminoethyl) amine (0.25 g, 1.8 mmol) (TREN) was added to the resulting yellow solution through a septum and the solution was adjusted to 48
Stir for hours. The solution was coagulated to dryness, and ethyl acetate (400m
l) collected in. N-hydroxysuccinimide was analyzed by thin layer chromatography (silica gel, CH ₂ Cl ₂ : CH ₃ OH = 90: 1).
The ethyl acetate layer was washed with water (1) until no longer detected by 0). The organic layer is dried over MgSO _4, and concentrated to dryness. The obtained solid was dissolved in CH ₃ OH (175 ml), a Sephadex LH-20 column (750 g) was added, and the mixture was eluted with CH ₃ OH. 72 hours later Tris-N- (2-
Aminoethyl- [8-hydroxyquinoline-2-carboxamido]) amine crystallized from the appropriate fraction tube as very fine white hairy crystals (0.75 g, 1.14 mmol, 63% yield). ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 2.96 (t, J = 6.
62Hz, 6H), 3.62 (q, J = 6.98Hz, 6H), 7.19 (d,
J = 7.72Hz, 3H), 7.46 (d, J = 8.09Hz, 3H), 7.57
(T, J = 7.72Hz, 3H), 8.15 (d, J = 8.82Hz, 3
H), 8.45 (d, J = 8.45Hz, 3H), 9.68 (t, J = 5.8)
8Hz, 3H) ¹³ C NMR (δ): 37.49, 53.69, 111.37, 117.40, 118.6.
5,129.16,129.30,136.31,137.46,147.42,153.4
5, 163.74 Mass spectrum (C ₃₆ H ₃₃ N ₇ O ₆ = 659.17 g / mol, M ⁺
H base peak): 660, 458, 244, 215, 171, 144, 93 Example 7 (N, N ', N "-tris (8-hydroxyquinoline-2-
Carboxamide) -1,4,7-Preparation of triazacyclononane) 2-carboxy-8 obtained in Example 4 in DMF (5.5 ml)
A mixture of -hydroxyquinoline (0.38 g, 1.98 mmol) and N-hydroxysuccinimide (0.23 g, 1.98 mmol) was stirred for 20 minutes and then 1- (3-
Dimethylaminopropyl) -3-ethylcarbodiimide.HCl (0.38 g, 1.98 mmol) was added. After 24 hours at room temperature 1,4,7-triazacyclononane (0.08 g, 0.62 mmol) in DMF (2.0 ml) [O. of Atkins et al.
rganic Synthesis, Vol.58, 1978, pp. 86-97, John ・
Manufactured by the method described in Willy and Sons, NY] was added and the reaction mixture was allowed to stir for 7 days.
DMF was removed by evaporation under vacuum and the residue was collected and combined with CH ₂ Cl ₂ (6
It was dissolved in 0 ml) and washed with water (3 × 60 ml). The organic layer was then dried over Na ₂ SO ₄ and evaporated to dryness under vacuum.
The crude residue was transferred to a Sephadex LH-20 column (bed 550
g) Chromatography on, eluting with methanol. N, N ', N "-tris (8-hydroxyquinoline-
2-Carboxamide) -1,4,7-triazacyclononane 0.06 g (15%) was recovered as a white solid. Mass spectrum (m / e): 643 (M + H) ⁺ .

Example ^{8 (N 1, N 4,} N 7 - ( tris - (8-hydroxy-2
Preparation of carboxamide) diethylenetriamine)) 2-Carboxy-8-hydroxyquinoline obtained in Example 4 (1.00 g, 5.3 mmol) was dissolved in dry DMF (100 ml). To this stirring solution was added N-hydroxysuccinimide (0.66 g, 5.7 mmol) and 1,3-dicyclohexylcarbodiimide (1.16 g, 5.7 mmol). The reaction vessel was sealed with a septum and stirred at room temperature for 24 hours. The precipitated 1,3-dicyclohexylurea byproduct was removed from the solution by filtration. Diethylenetriamine (0.18 g, 1.8 mmol) was added to the resulting yellow solution through a septum and the solution was stirred for 48 hours. The solution was concentrated to dryness and collected in ethyl acetate (400 ml). Thin layer chromatography of N-hydroxysuccinimide (silica gel, CH ₂ Cl ₂ : CH ₃ OH
= 85: 15) until the ethyl acetate layer was washed with water (1). The organic layer is dried over MgSO _4, and concentrated to dryness. The solid obtained is CH ₃ OH (275 ml)
Dissolved in, added to a Sephadex LH-20 column (750 g), and eluted with CH ₃ OH. The desired product was isolated by removing the solvent from the appropriate fraction tube (0.45 g, 0.729, as assessed by thin layer chromatography).
Mmol, yield 41%).

Example 9 (N ^1, N ⁷ - bis (t-butoxycarbonyl) Preparation of diethylenetriamine) diethylenetriamine in THF solution was cooled in an ice bath (100ml) (5.44g, 52.73 mmol) and triethylamine (15.97 g, 157.84 mmol) Was added. THF (500 ml)
BOC-ON (2- (t-butoxycarbonyloxyimino) -2-phenylacetonitrile) in (26.0 g, 105.6
(Mmol) solution was added dropwise to the above solution over 45 minutes. After stirring in an ice bath for 2 hours and room temperature for 1 hour, the solvent was removed in vacuo. Residual yellow / green oil was washed with ethyl acetate (350m
l) dissolved in cold (5 ° C) 5% aqueous solution of NaOH (5 x 2
It was extracted with 00 ml). The organic layer was dried over MgSO _4, and concentrated to an oil. The volume of this oil was 2% with 20% MeOH / 80% CH ₂ Cl ₂ .
Doubled and chromatographed on silica gel.
Thin layer chromatography of the fractions (10% MeOH / 90
% CH ₂ Cl ₂ , silica, ninhydrin). The fractions of the product, dried under vacuum until a clear gum N ^1, N ⁷ - to obtain a bis (t-butoxycarbonyl) diethylenetriamine (11.5 g). The gum was processed under high vacuum for a long time to give a white crystalline solid. Melting point: 69-71 ° C. ¹ H NMR (CDCl ₃ , TMS internal standard) δ: 1.44 (s, 18 H), 1.
65 (bs, 2H), 2.71 to 2.75 (t, J = 5.75Hz, 4H), 3.
19-3.25 (q, J = 5.70 Hz, 4H), 5.07 (bs, 1H) ¹³ C NMR (δ): 28.19, 40.21, 48.74, 79.15, 156.13 mass spectrum (C ₁₄ H ₂₉ N ₃ O ₄ = 303.4008 g / Mole
Direct chemical ionization, m / e): 304 ( M +, base _{peak), 248,192,173,130 IR (CDCl 3)} : 3454m, 2980s, 2933m, 1707s, 1505s, 145
5m, 1392m, 1367s, 1270m, 1249m, 1170s, 1046m Example 10 (N- (t-butoxycarbonyl) -4-nitro-L-
Preparation of phenylalanine-N-hydroxysuccinimidyl ester) Nt-BOC-para-nitro-L-phenylalanine (4.53 g, 14.59 mmol) was dissolved in ethyl acetate (150 ml), and N-hydroxysuccinimide ( 2.00
g, 17.39 mmol) and 1,3-dicyclohexylcarbodiimide (3.53 g, 17.11 mmol). The reaction flask was sealed with a septum and stirred at room temperature for 24 hours. The precipitated 1,3-dicyclohexylurea by-product was filtered off and the filtrate was concentrated to dryness. This white solid was recrystallized twice from 2-propanol (600 ml) to give N- (t-butoxycarbonyl) -4-nitro-L-phenylalanine-
N-hydroxysuccinimidyl ester was obtained as a white crystalline solid (4.92 g, 12.07 mmol, yield 82%). Melting point: 178-79 ° C. ¹ H NMR (CDCl ₃ , TMS internal standard) δ: 1.44 (s, 9H), 2.8
9 (s, 4H), 3.25 to 3.50 (m, 2H), 5.01 (bs, 1
H), 7.49 (d, J = 9.00Hz, 2H), 8.19 (d, J = 9.0
0 Hz, 2H) Mass spectrum (C ₁₈ H ₂₁ N ₃ O ₈ = 407.3792 g / mol for direct chemical ionization, m / e): 425 ((M + NH ₃ ) ⁺ , base peak), 408, 396, 352, 308 , 265,254,163,133 example 11 (N ⁴ - [1-oxo - (2-aminoethyl - (N-t-
Butoxycarbonyl)) - 2- ^{(4-nitrobenzyl)] - N 1, N 7} - bis (t-butoxycarbonyl) Preparation of diethylenetriamine) obtained in Example 10 N-(t-butoxycarbonyl) -4
-Nitro-L-phenylalanine-N-hydroxysuccinimidyl ester (3.0 g, 7.43 mmol) was dried on TH
Dissolved in F (150 ml) and added to this solution N ¹ , N
⁷ -Bis (t-butoxycarbonyl) diethylenetriamine (1.85 g, 6.09 mmol) was added. The flask was sealed with a septum and stirred at room temperature for 50 hours. The N-hydroxysuccinimide precipitate was filtered off and the filtrate was concentrated to dryness under vacuum to a clear gum. The gum was dissolved in methanol (25 ml) and the Sephadex LH-20 column (75
0 g bed weight, 100% methanol, 5 ml / min).
The desired material is thin layer chromatography (silica, 40%
Separation by observation with EtOAc / 60% CH ₂ Cl ₂ , Rf = 0.2). Appropriate fractions were concentrated to dryness under vacuum to give a clear gum. This material was dissolved in CH ₂ Cl ₂ (20 ml) and a silica column (100 g of 60 mesh silica,
95% CH ₂ Cl ₂ , 15 & MeOH, 10 ml / min). Again the appropriate fractions were collected by the same thin layer chromatography system to give a clear amorphous solid (melting point: 60-62
℃, 3.0 g, 5.04 mmol, yield 82%) ¹ H NMR (CDCl ₃ , TMS internal standard) δ: 1.38 (s, 9H), 1.4
2 (s, 18H), 3.30 (m, 4H), 3.28 (m, 2H), 3.35
(M, 4H), 4.98 (bs, 1H), 7.43 (d, J = 9.0Hz, 2
H), 8.17 (d, J = 9 Hz, 2H) mass spectrum (C ₂₈ H ₄₅ N ₅ O ₉ = 595.6916 g / mol,
Direct chemical ionization, NH ₃ , m / e): 596 ((M + H) ⁺ ,
Base peak), 540,496,484,440,384 Example 12 (N ⁴ - [1-oxo-2-aminoethyl-2- (4-nitrobenzyl)] - Preparation of diethylenetriamine acid trifluoroacetate) carried N ⁴ obtained in example 11 - [1-oxo-2-aminoethyl - (N-t-butoxycarbonyl)) - 2- ^{(4-nitrobenzyl)] - N 1, N 7} - bis (t-butoxycarbonyl ) Diethylenetriamine (1.0 g, 1.67 mmol) was dissolved in trifluoroacetic acid (15 ml) under argon atmosphere. The solution was stirred at room temperature for 1 hour and concentrated under vacuum to a pale yellow amorphous solid. This solid was redissolved in trifluoroacetic acid and the above procedure was repeated to completely remove the t-butoxycarbonyl protecting group. Solid (20m)
l) and lyophilized to give a pale yellow solid (1.21 g, 1.61 mmol, 95% yield). ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 3.10 to 3.70 (m,
10H), 4.63 (s, 1H), 7.57 (d, J = 9.0Hz, 2H),
7.98 (bs, 1H), 8.07 (bs, 1H), 8.22 (d, J = 9.0H
z, 2H), 8.42 (bs, 1H) mass spectrum (C ₂₁ H ₂₅ N ₅ O ₁₁ F ₁₂ = 751.436, free amine 295.1332, m / e): 296 (M ⁺ , free cation,
Base peak), 278,266,223,182,140,104 Example 13 (N ⁴ - [2- Amino-ethyl-2- (4-nitrobenzyl)] Preparation of diethylenetriamine tetrahydrochloride) In this embodiment, bifunctional tetrahydrochloride salt of TREN (N ⁴ - [2-
Aminoethyl-2- (4-nitrobenzyl)] diethylenetriamine tetrahydrochloride) was prepared. N ⁴ obtained in Example 12
-[1-Oxo-2-aminoethyl-2- (4-nitrobenzyl)] diethylenetriamine tritrifluoroacetate (1.15 g, 1.53 mmol) Dissolved in dry THF (25 ml) and placed in a condenser under argon atmosphere. Transferred to flask equipped with ice bath cooling. When the temperature of the solution reached the temperature of the ice bath, 1.0 M borane-THF (22 ml, 0.304 g, 36.8 mmol)
The solution was added through a septum and stirred at 0 ° C for 1 hour. The solution was then refluxed for 5.5 hours and cooled again to 0 ° C. Dry methanol (20 ml) was added to this solution. Since a large amount of gas is generated in this process, the process was performed with caution. When gas evolution from the solution ceased, anhydrous HCl was passed through the septum for 1 minute and then the solution was refluxed for 1 hour. The flask was left to cool to room temperature and concentrated under vacuum to a pale yellow solid. This material was dissolved in a minimum amount of methanol and added to Sephadex LH-20 (750g, 100% methanol, 3ml / min). Desired material is eluted first, fractions were evaluated by thin layer chromatography _{(silica, 40% NH 4 OH / 60} % absolute ethanol, ninhydrin). The appropriate fractions were collected and concentrated to dryness under vacuum N ⁴ - [2- Amino-ethyl-2- (4-nitrobenzyl)] diethylenetriamine tetrahydrochloride salt as a pale yellow amorphous solid (0.45 g, 1.05 mmol, yield 65
%). ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 2.21 to 2.46 (m,
2H), 2.78 to 3.01 (m, 8H), 3.15 to 3.25 (m, 2H),
4.03 (bs, 1H), 7.66 (d, J = 9.0Hz, 2H), 8.20
(D, J = 9.0 Hz, 2 H) Mass spectrum (C ₁₃ H ₂₃ N ₅ O ₂ .4HCl = 427.20, free amine 281.15, m / e): 282 (M ⁺ , free base, base peak), 265 , 252,239,146,126,114,104 example 14 (N ⁴ - [2- amino-ethyl-2- (4-nitrobenzyl)] tris-N-(8- hydroxyquinoline-2-carboxamido) diethylenetriamine Preparation) The 2-carboxy-8-hydroxyquinoline obtained in Example 4 (0.985 g, 5.21 mmol) was dissolved in dry THF (100 ml). To this stirred solution was added N-hydroxysuccinimide (0.650 g, 5.65 mmol) and 1,3-dicyclohexylcarbodiimide (1.150 g, 5.57 mmol). The reaction vessel was sealed with a septum and stirred at room temperature for 24 hours. The precipitated 1,3-dicyclohexylurea byproduct was removed from the solution by filtration. Example to the resulting yellow solution
13 obtained in N ⁴ - [2-aminoethyl-2- (4-nitrobenzyl) diethylenetriamine tetrahydrochloride (0.250 g, 0.58
Mmol) and triethylamine (1 ml) were added and the solution was stirred for 48 hours. The solution was concentrated to dryness and collected in ethyl acetate (400 ml). Thin layer chromatography of N-hydroxysuccinimide (silica, 90% CH ₂ Cl ₂ /10%
The ethyl acetate layer was washed with water (1) until no longer detected by (methanol). The organic layer is dried over MgSO _4, and concentrated to dryness. The solid obtained was dissolved in methanol (175 ml) and added to a Sephadex LH-20 column (750 g, bed weight, 100% methanol, 3 ml / min). Fractions were analyzed by the thin layer chromatography system, concentrated to dryness in vacuo The appropriate fractions were pooled N ⁴ - [2-aminoethyl-2- (4-nitrobenzyl)] tris-N-(8- Hydroxyquinoline-2
-Carboxamide) diethylenetriamine was obtained as a pale yellow amorphous powder (0.300 g, 0.37 mmol, yield 64).
%). ¹ H NMR (d ₆ -DMSO, TMS internal standard, 70 ° C.) δ: 2.98 to 3.12
(M, 2H), 3.28 to 3.37 (dd, J = 15.0Hz (twin), J
= 3.0Hz Ia ~ b, 2H), 3.49 ~ 3.63 (m, 4H), 3.64 ~
3.75 (m, 4H), 4.50 (s, 1H), 7.13 (d, J = 7.5H
z, 3H), 7.30 (d, J = 9.0Hz, 2H), 7.38 (d, J =
9Hz, 1H), 7.39 (d, J = 9Hz, 2H), 7.52 (t, J =
8.0Hz, 3H), 7.72 (d, J = 9.0Hz, 2H), 7.98 (d,
J = 9Hz, 1H), 8.12 (d, J = 9.0Hz, 2H), 8.34
(D, J = 8.5Hz, 1H), 8.39 (d, J = 9.0Hz, 2H),
9.29 (d, J = 10.5Hz, 1H), 9.60 (t, J = 6.0Hz, 2
H) Mass spectrum (C ₄₂ H ₃₈ N ₇ O ₆ NO ₂ = 794.8220 g / mol, direct chemical ionization, NH ₃ , m / e): 795 ((M + H) ⁺ , base peak), 765, 721, 706 , 607, 581, 458, 446, 3
36 ¹³ C NMR (many peaks show very close doublets due to rotomer barrier): 3
7.152, 37.412, 48.996, 53.673, 59.218, 111.090, 11
6.985, 118.37, 122.237, 128.798, 128.87, 129.099,
129.296, 136.125, 136.214, 137.061, 145.364, 146.9
52,147.301,147.362,153.344,163.230,163.627 Example 15 (N ⁴ - [2- Amino-ethyl-2- (4-aminobenzyl)] tris-N-(8- hydroxyquinoline-2-carboxamido) diethylenetriamine dihydrochloride Preparation of salt) N ⁴ obtained in Example 14 - [2-aminoethyl-2- (4-
Nitrobenzyl)] tris-N-(8- hydroxyquinoline-2-carboxamide) dihydrochloride diethylenetriamine (0.030 g, 0.03 mmol) was dissolved in CH ₃ CN (25ml). Add triethylamine (250μ, 18
1.5 mg, 1.78 mmol) and dry formic acid (100 μ, 122.
0 mg, 2.65 mmol) was added. Palladium on activated carbon (10 wt% palladium on carbon, 10 mg) was added to the solution and the suspension was then refluxed under an atmosphere of argon for 2.5 hours. The suspension was then left to return to room temperature. The palladium catalyst on carbon was removed by filtration and the filtrate was concentrated to dryness. The solid was suspended in 1N HCl (100 ml) and shaken for 5 min, concentrated the volume of the suspension to 5 ml, N ⁴ then isolated by lyophilization - [2-aminoethyl-2- (4 −
Aminobenzyl)] tris-N- (8-hydroxyquinoline-2-carboxamido) diethylenetriamine dihydrochloride was obtained as a light brown amorphous solid (0.024 g, 0.028).
Mmol, yield 95%).

Mass spectrum (C ₄₃ H ₃₈ N ₇ O ₆ NH ₂ = 764.8390, direct chemical ionization, NH ₃ , m / e): 765 ((M + H) ⁺ , base peak), 563, 458, 429, 383, 350 , 320,305 example 16 (N ⁴ - [2- amino-ethyl-2- (4-isothiocyanate benzyl)] tris-N-(8- hydroxyquinoline-2-carboxamide) preparation of diethylenetriamine hydrochloride) N ⁴ obtained in Example 15 - [2-aminoethyl-2- (4-
Aminobenzyl)] tris-N- (8-hydroxyquinoline-2-carboxamide) diethylenetriamine hydrochloride (35.0 mg, 0.041 mmol) was added to absolute ethanol (20 m
l). Add thiophosgene (6.4 μ
, 9.6 mg, 0.082 mmol) was added. The reaction flask was sealed with a septum and placed under an argon atmosphere. The solution was stirred at room temperature for 3.5 hours then the solvent was removed in vacuo. This provided the desired product as a light brown amorphous solid (0.030 g, 0.037 mmol, 91% yield).

Mass spectrum (C ₄₃ H ₃₈ O ₆ N ₇ · NCS · HCl = 806.8942 g / mol + HCl = 843.3551 g / mol, impact of positive fast atom, m / e): 807 (M ⁺ , base peak, parent cation ), 837, 765, 718, 458, 244, 215, 185, 144 Example 17 (Tris-N- (2-aminoethyl- [8-hydroxyquinoline-5-sulfonate-2-carboxamide])
Preparation of amine) 2-Carboxy-5-sulfonate-8-hydroxyquinoline (0.500 g, 1.85 mmol) in dry DMF (100 ml)
Dissolved in. To this stirred solution was added N-hydroxysuccinimide (0.225 g, 1.95 mmol) and 1,3-dicyclohexylcarbodiimide (0.405 g, 1.96 mmol). The reaction vessel was sealed with a septum and stirred at room temperature for 24 hours. The precipitated 1,3-dicyclohexylurea byproduct was removed from the solution by filtration. The resulting yellow solution was passed through a septum and tris (2-aminoethyl) amine (0.090 g,
0.619 mmol) was added and the solution was stirred at room temperature for 48 hours. The solution was concentrated to dryness and recrystallized twice from water to give the desired product as a microcrystalline white solid (0.365 g, 0.405 mmol, 65% yield). The desired product had improved solubility in aqueous solution. ¹ H NMR (d ₆ -DMSO, TMS internal standard) δ: 2.63 (m, 6 H),
2.92 (m, 6H), 7.15 (d, J = 9.0Hz, 3H), 8.03
(D, J = 9.1 Hz, 3H), 8.24 (d, J = 9.1 Hz, 3H) Example 18 Indium of (tris-N- (2-aminoethyl- [8-hydroxyquinolin-2-carboxamido]) amine Preparation of complex) Tris-N- (2-aminoethyl-) obtained in Example 6
[8-Hydroxyquinoline-2-carboxamido]) amine (0.100 g, 0.151 mmol) was dissolved in CH ₃ CN (about 225 ml) by heating to form a saturated reflux solution, and triethylamine (85 μ, 0.065 g, 0.604 mmol)
Was added. InCl ₃ (0.035 g, 0.159 mmol) was added to CH ₃ CN (3
5 ml) and then added dropwise to a refluxing solution of tris-N- (2-aminoethyl- [8-hydroxyquinoline-2-carboxamido]) amine / triethylamine. The solution was allowed to come to room temperature and the solvent was removed under vacuum to remove the indium complex of tris-N- (2-aminoethyl- [8-hydroxyquinoline-2-carboxamido]) amine in bright yellow-orange color. Obtained as a solid.

Mass spectrum (C ₃₆ H ₃₀ N ₇ O ₆ In = 770.9902 g / mol, impact of positive fast atom, m / e): 722 ((M +
H) ⁺ , base peak cation), 660, 558, 458, 36
9,329,299 Example 19 (N ⁴ - (2-aminoethyl-2- (4-isothiocyanatobenzyl)) tris-N-(8- hydroxyquinoline-2-carboxamide) conjugate of diethylenetriamine and monoclonal antibodies Preparation of mouse IgG ₁ monoclonal antibody specific for carcinoembryonic antigen (CEA) (Abbott Laboratories, North Chicago,
IL) 0.1M phosphate / 0.1M bicarbonate buffer (pH8.5)
Dialyzed into. The resulting solution was divided into 3 aliquots. Each aliquot, N ⁴ in DMSO - (2-aminoethyl-2- (4-isocyanate-benzyl)) tris -N-
A solution of (8-hydroxyquinoline-2-carboxamide) diethylenetriamine hydrochloride is added and the chelator: antibody ratio is 100: 1 for the first aliquot, 40: 1 for the second aliquot, and 3: 1 for the third aliquot. I made it 5: 1. After adding sufficient DMSO to make a homogeneous solution, all three aliquots were incubated at 37 ° C for 5 hours. The resulting solution was cooled to room temperature and centrifuged to remove any precipitated material. Each supernatant was then for 48 hours against a 0.05M DTPA solution in 0.05M citrate buffer,
It was then dialyzed for 4 days against multiple replacements of 0.05M citrate buffer and finally against 0.1M borate / 0.1M citrate buffer (pH 10.5).

Example 20 (N ⁴ - (2-aminoethyl-2- (4-isothiocyanatobenzyl)) tris-N-(8- hydroxyquinoline-2-carboxamide) conjugates with diethylenetriamine Indium-111 complex and CEA antibody The ability of the three antibody-chelating agent preparations obtained in Example 19 to specifically bind indium-111 was demonstrated by Meares et al., Anal. Biochem., 142, 68.
According to the method described in (1984), it was evaluated by performing DTPA challenge after the instant thin layer chromatography. Indium chloride-111 (500 μCi)
[New England Nuclear (New Englan
d Nuclear), Billerica, MA] of each of the three antibody-chelator conjugate solutions (100 μ)
And then incubated at room temperature for 2 hours. A 20 μ aliquot of each solution was then removed and mixed with a 0.05 M solution of DTPA (pH 6.0) (5 μ). The mixture was incubated at room temperature for 5 minutes, then silica gel impregnated fiberglass ITLC strips [Gelman Sciences, Ann Arbor,
MI] and developed with normal saline. Under these conditions, the indium-111 specifically bound to the chelator site of the antibody-chelator remains in the first position, but unbound indium-111 or the unbound indium-111 removed from the protein by the DTPA eluate. The weakly bound indium-111 migrates to the front of the solvent. Therefore, the activity of indium-111 that remains in the first position of the ITLC strip is an indicator of stable and specific binding to the antibody-bound chelator site. An untreated aliquot of the same anti-CEA antibody, i.e. an aliquot not bound to the chelating agent,
Served as a control. The results obtained are shown in Table 1.

These data show that a conjugate was formed between the isothiocyanate chelator of the invention and the anti-CEA antibody, and the degree of substitution was related to the chelator: antibody molar ratio used in the coupling reaction. Furthermore, these data indicate that these conjugates can specifically bind indium-111 under the attack of large excess of DTPA.

From the examples above, it will be apparent to those skilled in the art that the conjugates and methods of the invention are useful for imaging localized concentrations of antigen by external photoscanning as described by Goldenberg et al. Is. In such methods, the conjugate is introduced into the patient's body and the patient is scanned to determine the concentration of the conjugate. It will also be apparent that the conjugates of the invention can be used in in vitro diagnostics such as immunoassays and nucleic acid hybridization assays. In diagnostic methods such as sandwich hybridization methods, the conjugates of the present invention that include an indicator are useful for indicating the presence of an analyte. The conjugates and methods of the invention are also useful in therapeutic methods, in which case antibody-metal ion conjugates (where the metal ion emits cytotoxic radiation).
Is introduced into the patient's body to direct cytotoxic radiation to the tumor while minimizing toxic effects on healthy tissue. The chelating agents of the present invention are also useful in in vivo diagnostic methods for the administration or removal of both radioactive and non-radioactive metal ions.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C07D 213/65 C07D 213/65 215/26 215/26 215/48 215/48 401/14 255 401 / 14 255 C07K 7/04 8517-4H C07K 7/04 C09K 3/00 108 C09K 3/00 108

Claims (10)

(57) [Claims] 1. A general formula: (Where D is And a group selected from the group consisting of —CH ₂ —, R ₁ , R ₂ and R ₃ are the same or different, and are a hydrogen atom, a halogen atom,
Alkyl group C ₁ -C _3, a nitro group, a nitroso group, a sulfonate group, a sulfo trialkylammonium salt, a phenol group, a phosphate group, a carboxylic acid group of C ₁ -C _3, carboxamido group, a sulfonamido group, a phosphonic acid group And a group selected from the group consisting of a sulfate group, n is an integer of 3 or 4, A is a linear, cyclic or trigeminal triamine or tetraamine, wherein the nitrogen atom of each amine is Linked with alkane, cycloalkane or ortho-substituted phenyl ring substituents via nitrogen atoms between 2 and 4 carbon atoms and that all oxine units present are each bound to a single metal center Is positioned such that A can also be represented by the formula: Optionally substituted with a methylene carbon moiety by a group of the formula: wherein X is a nitro group in the meta or para position or a substrate-reactive residue and m is an integer from 0 to about 10. ) The compound shown by these. 2. In the above formula, A is and Is selected from the group consisting of: and The compound according to claim (1) represented by: 3. A general formula: (Where D is And a group selected from the group consisting of —CH ₂ —, R ₁ , R ₂ and R ₃ are the same or different, and are a hydrogen atom, a halogen atom,
Alkyl group C ₁ -C _3, a nitro group, a nitroso group, a sulfonate group, a sulfo trialkylammonium salt, a phenol group, a phosphate group, a carboxylic acid group of C ₁ -C _3, carboxamido group, a sulfonamido group, a phosphonic acid group And a group selected from the group consisting of a sulfate group, n is an integer of 3 or 4, A is a linear, cyclic or trigeminal triamine or tetraamine, wherein the nitrogen atom of each amine is Linked with alkane, cycloalkane or ortho-substituted phenyl ring substituents via nitrogen atoms between 2 and 4 carbon atoms and that all oxine units present are each bound to a single metal center Is positioned such that A can also be represented by the formula: Wherein Q is a substrate molecule, X is a nitro group or a substrate-reactive residue in the meta or para position, and m is an integer from 0 to about 10 and optionally at the methylene carbon moiety. Substrate conjugate with the compound according to claim (1), which is optionally substituted). 4. In the above formula, A is and The substrate conjugate according to claim (3), which is selected from the group consisting of: 5. In the above formula, Q is selected from the group consisting of proteins, glycoproteins, peptides, polyamino acids, lipids, carbohydrates, polysaccharides, nucleosides, nucleotides, nucleic acids, drugs, inhibitors and whole cells. The substrate conjugate according to claim (3). 6. A general formula: (Where D is And a group selected from the group consisting of —CH ₂ —, R ₁ , R ₂ and R ₃ are the same or different, and are a hydrogen atom, a halogen atom,
Alkyl group C ₁ -C _3, a nitro group, a nitroso group, a sulfonate group, a sulfo trialkylammonium salt, a phenol group, a phosphate group, a carboxylic acid group of C ₁ -C _3, carboxamido group, a sulfonamido group, a phosphonic acid group And a group selected from the group consisting of a sulfate group, n is an integer of 3 or 4, A is a linear, cyclic or trigeminal triamine or tetraamine, wherein the nitrogen atom of each amine is Linked with alkane, cycloalkane or ortho-substituted phenyl ring substituents via nitrogen atoms between 2 and 4 carbon atoms and that all oxine units present are each bound to a single metal center Is positioned such that A can also be represented by the formula: (Wherein Q is a substrate molecule, M is a metal ion, X is a nitro group in the meta or para position or a substrate-reactive residue,
m is an integer from 0 to about 10) and is optionally substituted with a methylene carbon moiety).
A substrate-metal ion conjugate with the compound according to claim (1). 7. In the above formula, A is and The substrate-metal ion conjugate according to claim (6), which is selected from the group consisting of: 8. A general formula: (Where D is And a group selected from the group consisting of —CH ₂ —, R ₁ , R ₂ and R ₃ are the same or different, and are a hydrogen atom, a halogen atom,
Alkyl group C ₁ -C _3, a nitro group, a nitroso group, a sulfonate group, a sulfo trialkylammonium salt, a phenol group, a phosphate group, a carboxylic acid group of C ₁ -C _3, carboxamido group, a sulfonamido group, a phosphonic acid group And a group selected from the group consisting of a sulfate group, n is an integer of 3 or 4, A is a linear, cyclic or trigeminal triamine or tetraamine, wherein the nitrogen atom of each amine is Linked with alkane, cycloalkane or ortho-substituted phenyl ring substituents via nitrogen atoms between 2 and 4 carbon atoms and that all oxine units present are each bound to a single metal center Where M is a metal ion and A also has the formula: Optionally substituted with a methylene carbon moiety by a group of the formula: wherein X is a nitro group in the meta or para position or a substrate-reactive residue and m is an integer from 0 to about 10. ), A metal ion conjugate with the compound according to claim (1). 9. The general formula: (In the formula, m is an integer of 0 to about 10, p is an integer of 0 or 1,
q is an integer of 0 or 1, r is an integer of 0 or 1, s is an integer of 2, 3 or 4, t is an integer of 2, 3 or 4, u
Is an integer of 0, 1, 2 or 3 and v is 0, 1, 2 or 3
, U + v is an integer of ₁ , 2 or 3 and S ₁ ,
_{S 2, S 3, S 4} , S 5 and S ₆ are the same or different, -H,
_{-OH, -CH 2 CO 2 H,} -CH 2 CH 2 CO 2 H, and (In the formula, y is an integer of 1 to about 5, S ₇ is an aryl group or C ₁
To C ₂₀ alkyl group) and the following formula and (Wherein S ₈ and S ₉ are the same or different and are C _{1 to} C ₂₀ alkyl groups or aryl groups), and a cyclic substituted or unsubstituted aromatic metal bond residue selected from the group A nitro group in the meta or para position or a substrate-reactive residue). 10. The general formula: (In the formula, m is an integer of 0 to about 10, s is an integer of 2, 3 or 4, t is an integer of 2, 3 or 4, u is an integer of 0, 1, 2 or 3, and v is 0, 1 An integer of 2 or 3, u + v is an integer of 1, 2 or 3, and X is a nitro group in the meta or para position or a substrate-reactive residue). The described compound.